Method of preparing a standard diagnostic gene transcript pattern

ABSTRACT

A method for preparing a gene transcript pattern probe kit characteristic of a disease or condition or a stage thereof in a prokaryotic or eukaryotic organism using mRNA which is differentially expressed in the disease or condition or stage as probes, methods of diagnosis using the method and kits for performing the same are disclosed.

The present invention relates to methods of preparing standarddiagnostic gene transcript patterns, probe kits for diagnosis andmethods of diagnosis using such kits.

There are numerous examples of diagnostic methods that include physical,anatomical and behavioural examination and/or biochemical, electrical,or electromagnetic studies and/or assays.

These diagnostic methods are well developed and are often efficientmeans to identify many pathological conditions. They are based on recentdevelopments and research as well as on the observations, experience andempirical data recorded by health-workers concerned with diseases ofhuman beings, other animals and plants for at least 6000 years.

Never before has the arsenal of diagnostic tools been greater than atpresent, but even so, incorrect diagnosis of ailments and otherconditions is still commonplace.

New diseases and conditions are found that may be related toenvironmental changes or mutations or other alterations in both theactive agents or organisms as well as in the organism that is exposed.In addition a number of old and new illegal substances used in sportsand by drug addicts do not have appropriate diagnostic tests for theirpresence.

Several conditions are not easily identified with the available methodsand/or the conclusive identification of a disease or condition may bereached too late for adequate corrective treatment.

Due to the extensive time often encountered in a complete diagnosticprocedure, incorrect antibiotic therapy is often started prematurely,before a conclusive diagnosis is reached. This medical practice canaggravate the serious development of bacterial strains resistant toantibiotics.

Thus, even though a great number of differential diagnostic methods havebeen developed, there is still a considerable number of closely relatedconditions, or combinations of conditions that resist quick, safe andsure identification at low cost.

Furthermore, a number of diagnostic methods depend on the injection offoreign fluids or other kinds of transfer of diagnostic aids onto orinto the organism under observation, or require biopsies. The removal ofsample tissue from parts of the organisms often not easily accessiblemay also have a detrimental effect on the identification process andhealing process itself.

It is known that certain diseases result in the elevated expression ofdifferent genes which may in some cases account for the pathogenicity ofthe disease or condition in question. Screening for the presence of aparticular transcript as an indication of the presence of disease hasthus been described (see for example Enderlin et al., FEBS Letters,(1997), 412, p 629-632). Methods for quantifying the levels of differenttranscripts by binding to cDNA derived from gene libraries, or bysequencing and electronic comparison to other libraries has beendescribed in for example Schena et al., (1996), PNAS USA, 93,p10614-10619; Schena et al., (1995), Science, 270, p467-470, Heller etal. (1997), PNAS USA, 94, p2150-2155 and International PatentApplication No. WO95/20681.

However, a quick and simple method using the characteristic pattern ofgene expression during disease or other conditions or stages thereof asa tool for diagnosis and/or prognosis, particularly a method which doesnot require any knowledge of the disease characteristics, the genesinvolved or their sequences has not been described.

It has now surprisingly been found that a simple method of diagnosis ofa disease or condition or stage thereof may be performed by thepreparation of a characteristic gene transcript pattern standard orfingerprint (standard diagnostic probe pattern) for thatdisease/condition followed by comparison of the transcript pattern of apatient or organism under study to that pattern. The standard isprepared by the identification of a number of specific and informativeprobes which serve as a set of markers for the disease or condition orstage thereof to be identified. These probes are bound to a solidsupport and then hybridized to mRNA, optionally reverse transcribedand/or amplified. The amount of the nucleic acid material which binds tothe different probes is assessed and together forms the transcriptpattern standard of that disease or condition or stage thereof.

Thus, in order to identify diseases, malaises or other conditions causedby other organisms, toxins, stress, ageing, environmental changes, etc,in human beings, animals, plants and all other living eukaryotic andprokaryotic organisms, a set of standard probe patterns of the amount oftranscript from one or more informative genes relative to a standard maybe developed, each such standard probe pattern being characteristic ofone ailment or condition and/or stage of such ailment. These standardprobe patterns are subsequently compared with a pattern of transcriptlevels, using the same probes, prepared from a recent sample of tissueor body fluid collected from a patient to be diagnosed, such patternsbeing specific to the present condition of the patient.

Usually the reaction to infections, toxins or deteriorations areaccompanied by changes in the level of activity in several or manygenes. These activity levels, that may either be relatively higher orlower, are together specific to the type of condition that isencountered. The normal activity and the altered activity may, to alarge extent, be measured by the amount of specific transcript or mRNAthat is present. Thus, standardised probes for analysis may be designedthat have patterns of activity that are characteristic for eachcondition or combination of conditions that is to be identified ordiagnosed. These standardised probes may be used to compare thestandardised probe pattern with transcript patterns from samples oftissue or body fluids prepared in a similar way and obtained from a livepatient or the organism to be studied.

In order to put the invention into practical use, two kinds ofsubstantially similarly developed diagnostic probes must be availablefor comparison.

1. A standard diagnostic probe pattern (SDPP) that is characteristic ofthe suspected ailment, developed from one or more organisms having thecondition or disease or stage thereof in question.and2. A patient specific probe pattern (PSPP) that is developed from arecently obtained sample of tissue or body fluid from a organism to bestudied.

To apply the invention to a specific condition, the pattern of a SDPPcharacteristic of the suspect ailment or stage thereof must have beendeveloped beforehand. In addition, a recent, well preserved sample oftissue or body fluid from the patient must be available to develop thePSPP, for comparison with one or several different SDPPs for the numberof ailments and their different stages that is suspected.

To design and develop the pattern for SDPPs, characteristic for oneailment, known techniques of isolation of mRNA, construction andamplification of cDNA and selection through differential hybridisationand differential display may be used.

Selected informative mRNA or cDNA probes from one or more patients thathave been conclusively diagnosed with the ailment in question areisolated and amplified. These SDPPs together will be used to compare ifthe PSPP is similar to the SDPP. Several such characteristic SDPPs maybe developed to represent different stages of the same ailment.

The pattern of such standard probes for a great number of ailments anddifferent stage of such ailments may be accumulated in databases and bemade available to laboratories on request.

Thus, viewed from one aspect the present invention provides a method ofpreparing a gene transcript pattern probe kit for diagnosing oridentifying a disease or condition or stage thereof in a prokaryotic oreukaryotic organism comprising at least the steps of:

-   -   a) isolating mRNA from the tissue, cells or body fluid of a        normal prokaryotic or eukaryotic organism (normal sample);    -   b) isolating mRNA from the corresponding tissue, cells or body        fluid of an organism of step a) having the disease or condition        of interest or a stage thereof (diseased sample);    -   c) separating the mRNA of steps a) and b), which may optionally        be reverse transcribed to cDNA, by a non-sequence based        separation technique;    -   d) selecting two or more mRNA or cDNA species which are present        at different levels in the normal and diseased samples;    -   e) isolating the mRNA or cDNA species identified in step d);    -   f) optionally reverse transcribing the mRNA of step d) or e) to        cDNA, unless this has previously been performed in step c); and    -   g) immobilizing the mRNA or cDNA probes of step e) or f) on one        or more solid supports.

As used herein the disease or condition may be any condition, ailment,disease or reaction that leads to the relative increase or decrease inthe activity of informative genes of any or all eukaryotic orprokaryotic organisms regardless of whether these changes have beencaused by the influence of bacteria, virus, prions, parasites, fungi,radiation, natural or artificial toxins, drugs or allergens, includingmental conditions due to stress, neurosis, psychosis or deteriorationsdue to the ageing of the organism, and conditions or diseases of unknowncause.

Such diseases include those which result in metabolic or physiologicalchanges, such as fever-associated diseases such as influenza or malaria.Other diseases which may be detected include for example yellow fever,sexually transmitted diseases such as gonorrhea, fibromyalgia,candida-related complex, cancer (for example of the stomach, lung,breast, prostate gland, bowel, skin etc), Alzheimer's disease, diseasecaused by retroviruses such as HIV, senile dementia, multiple schlerosisand Creutzfeldt-Jakob disease to mention a few.

The invention may also be used to identify patients with psychiatric orpsychosomatic diseases such as schizophrenia and eating disorders. Ofparticular importance is the use of this method to detect diseases, orstages thereof, which are not readily detectable by known diagnosticmethods, such as HIV which is generally not detectable using knowntechniques 1 to 4 months following infection. Conditions which may beidentified include for example drug abuse, such as the use of narcotics,alcohol, steroids or performance enhancing drugs. The diagnostic methodmay be used alone as an alternative to other diagnostic techniques or inaddition to such techniques. For example, methods of the invention maybe used as an alternative or additive diagnostic measure to diagnosisusing imaging techniques such as Magnetic Resonance Imagine (MRI),ultrasound imaging, nuclear imaging or X-ray imaging, for example in theidentification and/or diagnosis of tumours.

“Stages” thereof refer to different stages of the disease or conditionwhich may or may not exhibit particular physiological or metabolicchanges, but does exhibit changes at the genetic level which may bedetected as altered gene expression. It will be appreciated that duringthe course of a disease or condition the expression of differenttranscripts may vary. Thus at different stages, altered expression maynot be exhibited for particular transcripts compared to “normal”samples. However, informative probes are selected from those transcriptswhich exhibit altered expression at one or more stages through thecourse of the disease and which together with information relating tothe level of other transcripts can be used to provide a characteristicpattern which is indicative of a particular stage of the disease. Suchinformative probes may be identified by comparing the transcripts ofnormal samples with transcripts of diseased samples from one or morestages of the disease or condition, or by comparing different stages ofthe disease or condition to one another, according to the abovedescribed method.

As used herein the prokaryotic or eukaryotic organism may be anyeukaryotic organisms such as human beings, other mammals and animals,birds, insects, fish and plants, and any prokaryotic organism such as abacteria.

As used herein the “tissue” may be a tissue obtained during surgery, forexample by biopsy, or by other means. “Cells” include cells isolatedfrom tissues or body fluids or body waste or in the case of prokaryoticorganisms, the organism itself. “Body fluids” include urine, blood,semen etc. It will however be appreciated that the method of preparingthe standard transcription pattern and method of diagnosis of theinvention is also applicable to use on living parts of eukaryoticorganisms such as cell lines and organ cultures and explants.

“Normal” as used herein refers to organisms or samples which are usedfor comparative purposes. Preferably, these are “normal” in the sensethat they do not exhibit any indication of, or are believed to have, anydisease or condition that would affect gene expression, particularly inrespect of the disease for which they are to be used as the standard.However, it will be appreciated that different stages of a disease orcondition may be compared and in such cases, the “normal” samplecorresponds to the earlier stage of the disease or condition. “Disease”samples and organisms include samples and organisms suffering from anailment or particular condition and are those known to have, or whichexhibit, the disease or condition or stage thereof under study.

“Complementary strands” is used in the conventional sense to refer tostrands of DNA which are complementary at each base to the template cDNAfrom which they are derived. “cDNA” as referred to herein includes firststrand cDNA produced by reverse transcription of RNA and complementarystrands to the first strand cDNA, namely second strand cDNA.

“Different levels” of the nucleic acids species refers to quantitativeor qualitative differences suggestive of differential expression. A“probe” may be one or more nucleic acid molecules which may be the sameor different (ie. a mixture) but which as a whole are differentiallyexpressed in the normal and diseased samples. When different stages ofthe disease/condition are investigated, differential expression of thetranscripts corresponding to the probes should be exhibited eitherrelative to an undiseased sample or relative to a different stage of thedisease/condition. Generally such probes are cDNA reverse transcribedfrom mRNA, or its complementary strands, although the mRNA itself mayalso be used. This could for example be achieved using a DNA fragment asa template for the probe by insertion in a vector behind a T3 or similarpromoter. It will be appreciated however that this nucleic acid materialmay be modified without affecting performance of the invention providinghybridization to sample nucleic acid molecules is still possible. Thusthe nucleic acid molecules referred to herein, such as mRNA and cDNA,includes molecules which are modified (e.g. methylated) or which includemodified or non-natural bases which may be used in the preparation ofthe cDNA or during amplification. Similar considerations apply to anynucleic acid molecules described herein. Thus for example, thetranscripts, present in the samples, may be in the form of RNA oraltered to modified forms of RNA and/or into modified or unmodifiedforms of DNA and/or in the form of, primers, antibodies or othermolecules that recognize and bind to target probes, particularly byspecific hybridisation to the target probes.

“Assessing” as used herein refers to both quantitative and qualitativeassessment which may be determined in absolute or relative terms. Thecharacteristic “pattern” created by this technique refers to informationwhich for example may be represented in tabular or graphical form andconveys information about the signal associated with two or more probes.

As used herein, reference to “corresponding” tissues etc. refers topreferably the same tissue, cells or fluid, but also includes tissue,cells or fluid which are sufficiently similar for the purposes ofpreparing the standard. When used in reference to genes “corresponding”to the probes, this refers to genes which are related by sequence (whichmay be complementary) to the probes although the probes may reflectdifferent splicing products of expression. Thus two separate probes maybe developed for a single disease which are transcribed from the samegene but reflect different splicing events. However the use of probeswhich reflect altered gene expression of two or more distinct genes ispreferred.

This invention relates to both a diagnostic principle and a method ofidentifying diseases, malaises and syndromes in any eukaryotic orprokaryotic organism as well as the associated method for the design anddevelopment of diagnostic probes to be used in the relative measurementsnecessary to reach specific diagnosis or to identify relevantconditions.

The invention is a quick and precise method for the diagnosis of anydisease or condition that leads to alterations in the activity of genesin a pattern which is specific to any particular condition of theorganism under observation.

The ability to design diagnostic standard probes for the identificationof traditional conditions, that at present are hard to identify, as wellas the ability to quickly adapt the design to prepare new probes for theidentification of new conditions that may appear, as soon as they areidentified, will therefore be of great value.

From the very early stages of diseases caused by infections, toxicsubstances, ageing or other conditions changing the quality of life ofliving eukaryotic organisms, the whole organism responds to the changedcondition. The response occurs, throughout the organism, even if only aminor part of the organism appears to be affected. The response lastsuntil the condition is healed or until the death of the affectedorganism

The advantages of the invention are of both primary and secondarynature. Samples of tissue or body fluids may be obtained from parts ofthe organism that are not affected by the condition under observation.One sample will suffice for a complete identification, thus greatreductions in costs, time and inconvenience may result by avoidinghospitalization during the normal extensive range of diagnostic testsperformed on human and other animal patients.

No foreign substances need be introduced onto or into the organism underobservation in order to aid in the identification of the condition, thusthe invention will reduce the risk of anaphylactic reactions to suchinduced diagnostic substances.

The invention has the potential to detect most diseases and syndromes ofsomatic, psycho-somatic and mental character as well as detectingdeterioration due to ageing of the organism. In addition the method maybe used to detect the organism's reactions to toxic substances,radiation, pesticides, antibiotics, drugs, allergens and combinations ofseveral such conditions.

The invention will furthermore make it possible to detect diseases orundesirable conditions in an organism at very early stages, even yearsbefore other subjective or objective symptoms may appear.

Even in cases where the patient dies from a hitherto unidentifiedcondition and the cause of death is not established until a forensicpost-mortem examination has been performed, the principle will be ofvalue. If, in the attempt to diagnose the patient prior to death, aseries of patient specific probe patterns were developed, these probesmay be used for the design of new standard probe patterns that may beused to diagnose later occurrences of similar conditions.

The analytical instruments and equipment necessary to make use of theinvention is readily available in laboratories engaged in standardbiochemical and bio-technological work.

To begin the preparation of the gene transcript pattern probe kit, mRNAis extracted from the tissues, cells or body fluid according to knowntechniques (see for example Sambrook et. al. (1989), Molecular Cloning:A laboratory manual, 2nd Ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.) from a normal individual or organism.

mRNA is also extracted, preferably from the same body part of acorresponding individual or organism which has the disease or conditionfor which the standard diagnostic pattern is to be created. Owing to thedifficulties in working with RNA, the RNA is preferably reversetranscribed at this stage to form first strand cDNA, although this maybe performed after separation and identification of transcripts ofinterest if cDNA probes are to be generated. Cloning of the cDNA orselection from, or using, a cDNA library is not however necessary inthis or other methods of the invention.

Preferably, the complementary strands of the first strand cDNAs aresynthesized, ie. second strand cDNAs, but this will depend on whichstrands are proposed to be used as the probes and the nature of thenucleic acid molecules in the sample to be probed during the diagnosismethod. The second strand cDNA strands are preferably used to probe cDNAstrands which have been produced by reverse transcription of the samplemRNA.

Preferably the cDNA strands are amplified by known amplificationtechniques such as the polymerase chain reaction (PCR) by the use ofappropriate primers. Alternatively, the cDNA strands may be cloned witha vector, used to transform a bacteria such as E. coli which may then begrown to multiply the nucleic acid molecules. When the sequence of thecDNAs are not known, primers may be directed to regions of the nucleicacid molecules which have been introduced. Thus for example, asdescribed in the Examples herein, adapters may be ligated to the cDNAmolecules and primers directed to these portions for amplification ofthe cDNA molecules. Alternatively, in the case of eukaryotic samples,advantage may be taken of the polyA tail and cap of the RNA to prepareappropriate primers.

Separation of the normal and diseased sample mRNA or cDNA is performedseparately on each sample by non-sequence based separation techniqueswhich are any suitable techniques which allows discrimination betweentranscripts or their corresponding cDNAs without involving the use ofsequence information of particular transcripts to discriminate betweenthe different transcripts/cDNA. This does not however exclude thepossibility of for example using probes carrying labels to producesignals on the transcripts if the probes are directed to sequencescommon on all or most of the transcripts which are not used for thepurpose of, discriminating transcripts. Thus conveniently, mRNA or cDNAmay be separated by electrophoretic separation on an agarose orpolyacrylamide gel or a similar gel appropriate for the separation ofthe nucleic acid molecules, Alternatively, the products may be separatedby gas chromatography or HPLC or similar techniques. (Sequence basedseparation techniques, which are excluded, include for example capturewith probes directed to different sequences by hybridization orsequencing itself.)

Such methods offer the advantage that probes to be used in the methodsof the invention are identified and selected from the entire populationof transcripts or cDNA since no selection is made on the basis of theirsequence before separation, e.g. by hybridization to immobilized nucleicacid from control samples. Thus, the identification of the transcriptsis not biased towards the selection of particular transcripts from asubset of total transcripts.

To allow comparison between the samples, separation of thetranscripts/cDNA should be performed on the normal and diseased samplesas simultaneously as possible, e.g. consecutive runs or on the same gel.

To identify the different transcripts or cDNA, it is necessary toidentify a signal corresponding to each transcript/cDNA. Convenientlythis may be achieved by the use of a radioactive or other label whichmay be incorporated during cDNA production or during amplification.

Appropriate labels are those which directly or indirectly allowdetection or measurement of the presence of the transcripts/cDNA. Suchlabels include for example radiolabels, chemical labels, for examplechromophores or fluorophores (e.g. dyes such as fluorescein andrhodamine), or reagents of high electron density such as ferritin,haemocyanin or colloidal gold. Alternatively, the label may be anenzyme, for example peroxidase or alkaline phosphatase, wherein thepresence of the enzyme is visualized by its interaction with a suitableentity, for example a substrate. The label may also form part of asignalling pair wherein the other member of the pair is found on, or inclose proximity to, the target probe to which the transcript/cDNA binds,for example, a fluorescent compound and a quench fluorescent substratemay be used. A label may also be provided on a different entity, such asan antibody, which recognizes a peptide moiety attached to thetranscripts/cDNA, for example attached to a base used during synthesisor amplification.

A signal may be achieved by the introduction of a label before, duringor after the separation step. Thus for example, a gel on which thetranscripts have been separated could be probed with labelled polyToligonucleotides, or cDNA could be probed with labelled polyAoligonucleotides or probed with labelled oligonucleotides directed to asequence which is introduced by ligation and/or amplification.Alternatively, the presence of transcripts could be identified by otherphysical properties, such as their absorbance, if techniques such as gaschromatography or HPLC are employed for separation.

Depending on the technique used for separation, signals for differenttranscripts or their cDNA may overlap and not be fully resolved. Whilstprobes may be produced which contain a mixture of transcripts or theircDNA (providing the mix as a whole exhibits differential expression innormal and diseased samples), optionally, the mixture oftranscripts/cDNA may be extracted and subjected to repeated oralternative separation techniques to isolate a smaller population oftranscripts/cDNAs which exhibits the altered expression.

Nucleic acid species exhibiting differential expression in normal versusdiseased samples, or in at least one stage of said disease, areidentified. This requires comparison between the signals produced by thenormal and diseased samples. Transcripts/cDNAs of interest are thosewhich are differentially expressed in the different samples as exhibitedby different amounts of signal. This may correspond to a species presentin the diseased sample and not in the normal sample or vice versa. Inthis way both gene expression which is turned on and gene expressionwhich is turned off is reflected. This offers significant advantagesover prior art methods in which altered gene expression is identifiedrelative to normal samples (in which cDNA from normal libraries is usedas the hybridization template to which sample mRNA is bound and therelative difference between “normal” and “diseased” samples aremeasured). The method of the invention thus can identify genes which arenot constitutively expressed or are expressed only at very low levels innormal samples, but are activated in the disease/condition, or at leastone stage thereof, in question.

Transcripts/cDNA which reflect a variation in the extent of expressionmay be used, but it is preferable to use those species which reflectmarked differences, e.g. absence or presence of a transcript/cDNA.

Once identified, the mRNA or cDNA species (which may be a mixture ofmolecules having different sequences) are isolated. To prepare astandard diagnostic pattern, two or more species (probes) which reflectaltered gene expression in at least one stage of said disease orcondition may be isolated. Whilst in some cases only two probes may besufficient to produce a standard diagnostic pattern for a particularcondition or disease or stage thereof, it will be appreciated thatincreasing the number of probes will prevent the possibility ofmisdiagnosis by comparison to other diseases which could similarly alterthe expression of the particular genes in question. Thus preferablybetween 2 and 1000 probe species are selected for isolation, especiallypreferably between 10 and 500, particularly preferably 50 and 100, forexample 70 probe species. These probes reflect genes which have alteredexpression in the diseases or conditions in question, or particularstages thereof are considered “informative” for that particular diseasein the organism under study and only those probes having thisinformative property are selected.

Isolation may be by selection of appropriate fractions if for exampleseparation was performed on a column, or by physical removal from theseparation matrix, for example excision of gel slices containing thenucleic acid species of interest. The nucleic acid molecules containedtherein are then isolated and purified if necessary for the subsequentsteps, preferably with amplification.

In instances in which the transcripts themselves have been separated andisolated, these may then be converted to cDNA, preferably withamplification.

The mRNA or cDNA probe species produced according to the above methodare then each immobilized onto a solid support to produce the disease'sor condition's probe kit or gene transcript pattern (or fingerprint)probe kit. Numerous solid supports suitable as immobilizing moieties fornucleic acid molecules are well known in the art and widely described inthe literature and generally speaking, the solid support may be any ofthe well-known supports or matrices which are currently widely used orproposed for immobilization, separation etc. in chemical or biochemicalprocedures. Thus for example, the immobilizing moieties may take theform of particles, sheets, gels, filters, membranes, microfibre strips,tubes or plates, fibres or capillaries, made for example of a polymericmaterial e.g. agarose, cellulose, alginate, teflon, latex orpolystyrene. Particulate materials, e.g. beads, are generally preferred.Conveniently, the immobilizing moiety may comprise magnetic particles,such as superparamagnetic particles.

Attachment of the nucleic acid molecules to the solid support may beperformed directly or indirectly. For example if a filter is used,attachment may be performed by UV-induced crosslinking. Alternatively,attachment may be performed indirectly by the use of an attachmentmoiety carried on the nucleic acid molecules and/or solid support. Thusfor example, a pair of affinity binding partners may be used, such asavidin, streptavidin or biotin, DNA or DNA binding protein (e.g. eitherthe lac I repressor protein or the lac operator sequence to which itbinds), antibodies (which may be mono- or polyclonal), antibodyfragments or the epitopes or haptens of antibodies. In these cases, onepartner of the binding pair is attached to (or is inherently part of)the solid support and the other partner is attached to (or is inherentlypart of) the nucleic acid molecules.

Attachment of appropriate functional groups to the solid support may beperformed by methods well known in the art, which include for example,attachment through hydroxyl, carboxyl, aldehyde or amino groups whichmay be provided by treating the solid support to provide suitablesurface coatings. Attachment of appropriate functional groups to thenucleic acid molecules of the invention may be performed by ligation orintroduced during synthesis or amplification, for example using primerscarrying an appropriate moiety, such as biotin or a particular sequencefor capture.

The individual probes form modules of the kit and may be present on oneor more solid supports. The solid support of the different modules areconveniently physically associated although the signals of each probemust be separately determinable. Thus for example, plates with multiplewells may be used as the solid support with different probes in thedifferent wells, or regions of a solid support may comprise thedifferent modules, for example the different mRNA or cDNA probes may bebound to a filter at discrete sites.

Thus in a preferred aspect the present invention provides a method ofpreparing a gene transcript pattern probe kit for diagnosing oridentifying a disease or condition or stage thereof in a prokaryotic oreukaryotic organism comprising at least the steps of:

-   -   a) isolating mRNA from the tissue, cells or body fluid of a        normal prokaryotic or eukaryotic organism (normal sample);    -   b) isolating mRNA from the corresponding tissue, cells or body        fluid of an organism of step a) having the disease or condition        of interest or a stage thereof (diseased sample);    -   c) reverse transcribing the mRNA of steps a) and b) to cDNA;    -   d) optionally amplifying said strands, optionally incorporating        a label into said strands;    -   e) separating the cDNA of step d) by a non-sequence based        separation technique;    -   f) selecting two or more cDNA species which are present at        different levels in the normal and diseased samples;    -   g) isolating the cDNA species identified in step f); and    -   h) immobilizing the cDNA probes of, step g) on one or more solid        supports.

The gene transcript pattern probe kits prepared according to the abovemethod for diagnosing or identifying a particular disease/condition orstage thereof in a particular individual/organism or for preparing astandard diagnostic gene transcript pattern, form further aspects of theinvention.

Thus viewed from a further aspect the present invention provides a genetranscript pattern probe kit for diagnosing, identifying or preparing astandard diagnostic gene transcript pattern of a disease or condition orstage thereof in a eukaryotic or prokaryotic organism comprising atleast the following:

-   -   a) one or more solid supports carrying two or more probe species        according to the invention corresponding to transcripts which        reflect gene expression of one or more selected genes        characteristic in the condition or disease or stage thereof in        the organism under investigation.

Optionally the kit may also contain information relating to the signalsgenerated by normal or diseased samples, standardizing materials, e.g.mRNA or cDNA from normal and/or diseased samples for comparativepurposes, labels for incorporation into cDNA, adapters for introducingnucleic acid sequences for amplification purposes, primers foramplification and/or appropriate enzymes, buffers and solutions.

The use of such kits to prepare a standard diagnostic gene transcriptpattern forms a further aspect of the invention.

In a yet further aspect, the present invention provides

a method of preparing a standard diagnostic gene transcript patterncharacteristic of a disease or condition or stage thereof in aprokaryotic or eukaryotic organism comprising at least the steps of

-   -   a) isolating mRNA from the tissue, cells or body fluid of said        organism having the disease or condition or stage thereof, which        may optionally be reverse transcribed to cDNA,    -   b) hybridizing the mRNA or cDNA of step a) to the mRNA or cDNA        probes on a kit according to the invention specific for said        disease or condition or stage thereof in an organism        corresponding to the organism under investigation; and    -   c) assessing the amount of mRNA or cDNA hybridizing to each of        said probes on said solid support(s) to produce a characteristic        pattern reflecting gene expression in the sample with the        disease, condition onstage thereof of one or more selected genes        corresponding to the probes.

To produce the standard diagnostic gene transcript pattern orfingerprint for a particular disease or condition or stage thereof, theabove produced probe kit is used to probe mRNA or cDNA of a diseasedsample to give a signal for hybridization to each particular probespecies bound to the solid support, ie. the kit modules. A standardcontrol gene transcript pattern may be prepared if desired using mRNAfrom a normal sample. Thus, total mRNA isolated in the same manner asdescribed above, or its cDNA (depending on the complementarity of theprobe species on the probe kit module) of a diseased sample(corresponding to the disease to which the probes aredirected)(optionally also a normal sample) is hybridized underappropriate conditions to the probe species on the probe kit modules.When both samples are probed, this may be performed consecutively on thesame probe kit modules, by simultaneously hydridizing to the modules ofa corresponding probe kit.

To obtain an indication of the number of transcripts/cDNA moleculeswhich become bound to the probe kit modules, the signal produced whenthe transcripts hybridize is detected (e.g. by detection of doublestranded nucleic acid molecules or detection of the number of moleculeswhich become bound, after removing unbound molecules, e.g. by washing).In the latter case, preferably labelled mRNA/cDNA molecules are used asthe sample, for example by incorporating radiolabelled bases duringreverse transcription, the preparation of complementary cDNA strands oramplification. The amount of signal is then assessed for each probe kitmodule. The assessment may be quantitative or qualitative and may bebased on binding of a single transcript species to each probe, acombination of transcripts, or representative forms of the transcriptssuch as cDNA or modified nucleic acid molecules as described above. Itwill be appreciated that quantitative results will provide furtherinformation for the transcript fingerprint of the disease which iscompiled. This data may be expressed as absolute values or may bedetermined relative to a particular standard or probe module result. Thevalue of the signal from the normal sample may be subtracted from thesignal from the disease sample, where the former is determined, althoughpreferably results from test samples are compared to the unstandardizeddisease fingerprint which is produced.

Furthermore it will be appreciated that the standard diagnostic genepattern transcript may be prepared using the results of one or morediseased and/or normal samples to identify probes for the probe kit, andone or more disease samples (and normal samples if used) may be used toperform the hybridization step to obtain the standard diagnostic genetranscript pattern.

The use of such kits and standard diagnostic gene transcript patternsfor the purpose of identification or diagnosis of a particular diseaseor condition or stage thereof in a particular organism forms a furtheraspect of the invention.

Once a standard diagnostic fingerprint or pattern has been determinedfor a particular disease or condition using the selected probe species,this information can be used to identify the presence, absence or extentof that disease or condition in a different organism or individual.

Thus viewed from a further aspect the present invention provides amethod of diagnosing or identifying a disease or condition or stagethereof in a prokaryotic or eukaryotic organism, comprising the stepsof:

-   -   a) isolating mRNA from the tissue, cells or body fluid of said        organism, which may optionally be reverse transcribed to cDNA;    -   b) hybridizing the mRNA or cDNA of step a) to a kit of the        invention specific for said disease or condition or stage        thereof in an organism corresponding to the organism under        investigation;    -   c) assessing the amount of mRNA or cDNA hybridizing to each of        said probes on said solid supports to produce a characteristic        pattern reflecting gene expression of one or more selected genes        corresponding to the probes;    -   d) comparing said pattern to a standard diagnostic pattern        prepared according to the method of the invention using a sample        from an organism corresponding to the organism under        investigation having said disease or condition or stage thereof        under investigation to determine the degree of correlation        indicative of the presence of said disease or condition or a        stage thereof in the organism under investigation.

The degree of correlation which is required to confirm the presence,absence or extent of a disease or condition necessarily takes intoaccount the range of values which are obtained for normal and diseasedsamples. Although this can be established by obtaining standarddeviations for several representative samples binding to the probes todevelop the standard, it will be appreciated that single samples may besufficient to generate the standard pattern to identify a disease if thetest sample exhibits close enough correlation to that standard.

The diagnostic method may be used to identify, quantify or diagnose adisease, condition or ailment or its stage or progression, for examplecancer in humans and bovine spongiform encephalopathy in cattle. Themethods of the invention may also be used to monitor the condition/stateof plants, for example to monitor the effects of pollution, or in thecase of prokaryotic organisms, to monitor their state during suchprocesses as fermentation or sewage processing.

Owing to the effects that certain exogenous factors or diseases exert onall parts of body, ie. the effects on gene expression are not isolatedto the areas of apparent disease, and thus body parts distant from thesite of interest, e.g. a tumour, may be analysed. Thus samples may beobtained for testing in a non-invasive manner, such as for example abody fluid, such as blood. Since samples may originate from differentparts of the body which may exhibit some differences in theirtranscription products, samples used to prepare the standard and testsamples should preferably be matched. In the diagnosis methods of theinvention three different samples are concerned which could be derivedfrom different sources. These are the samples used to prepare theprobes, the disease sample used to prepare the standard diagnosispattern and the test sample. Preferably all samples are derived fromcomparable sources, but especially preferably at least the diseasesample used to prepare the standard pattern and the test sample arederived from a corresponding source.

The following examples are given by way of illustration only in whichthe Figures referred to are as follows:

FIG. 1 shows a standard diagnostic gene expression pattern in graphicalform for a disease in Arabidopsis; and

FIG. 2 shows the extent of binding of cDNA isolated from Picea abieschallenged with different types of stress to 7 different probes.

EXAMPLE 1 Diagnosis of Alzheimer Syndrome

A blood sample is collected from a patient suspected of suffering fromthe ailment. The sample is immediately preserved in liquid nitrogen toprevent degradation of the mRNA of the sample.

The sample is transferred for analysis, the mRNA is converted to cDNA,amplified by PCR (Polymerase Chain Reaction) and labelled with suitableradioactive nucleotides.

The labelled cDNA is hybridized to several diagnostic DNA probes thatare immobilized on a filter.

The radioactive signals from the PSPP filter are quantified using anInstant Imager and the relative signal value from each of the differentprobes is determined.

The relative values from the different probes will together create apattern that is specific for the patient's ailment, the PSPP.

This specific pattern for the patient is compared to the SDPPscharacteristic of Alzheimer syndrome in different stages. If thepatterns of the SDPP and the PSPP coincide, the ailment and its stage isdiagnosed with great certainty.

If the patterns do not coincide, the ailment may be ruled out and thesame PSPP may be compared manually or automatically to any number ofavailable SDPPs until a match is found.

EXAMPLE 2 Diagnosis of Senile Dementia

The patient specific sample is collected, preserved, the mRNA isconverted to cDNA, amplified and labelled through the same procedure asdescribed in Example 1.

The comparative hybridization is performed with a different set of SDPPsthat have been developed to distinguish between different kinds andstages of senile dementia.

EXAMPLE 3 Broad Spectrum Health Status

The patient specific sample is collected, preserved, the mRNA isconverted to cDNA, amplified and labelled through the same procedure asdescribed in Example 1.

Primers to detect a set of transcripts are used for labelling.

The labelled samples are separated through gel electrophoresis and allthe DNA fragments are detected and quantified.

The resulting pattern specific to the patient PSPP is subsequentlycompared to SDPPs of a number of different diseases. For every mismatchencountered the associated ailment may be ruled out.

EXAMPLE 4 Diagnosis of a Disease/Condition in Arabidopsis

The diagnosis may be divided in two steps:

-   -   Development of a reproducible expression pattern (fingerprint)        typical for the disease/condition to be diagnosed.        -   Applying the developed method to a sample from an            undiagnosed patient or organism and comparing the expression            pattern to patterns developed for the disease/condition.

4.1 Development of a Reproducible Expression Pattern Typical for theDisease/Condition to be Diagnosed.

4.1.1 Extraction of Total RNA from Leaves of Arabidopsis

Samples of 15 g of leaves each from plants with a diagnosed disease andfrom healthy plants are collected and frozen immediately in liquidnitrogen. Each sample is divided into two aliquots.

The leaves from one of the two aliquots from each sample are ground witha mortal and pestle in liquid nitrogen to a fine powder. The secondaliquot of each sample is stored at −70° C.

The ground leaves are immediately transferred to a 500 ml beaker with150 ml grinding buffer (0.18M Tris-HCl, pH 8.2, 0.09M LiCl, 4.5 mM EDTA,1% (w/v) Sodium dodecyl Sulfate (SDS) plus 50 ml TLE-equilibrated phenol(TLE=0.2M Tris-HCl, pH 8.2, 0.1M LiC1, 5 mM EDTA)).

The mixture is homogenized with a polytron for 2 min at moderate speed(setting 5-6). 50 ml chloroform is added and mixed into the homogenateusing the Polytron. The slurry is poured into a centrifuge bottle andheated for 20 min at 50° C. This is then centrifuged for 20 min at 17700×g, 4° C.

After centrifugation, as much of the aqueous layer as possible isremoved without disturbing the interface and in a new centrifuge bottlethis phase is mixed with 50 ml TLE-equilibrated phenol by shaking. 50 mlchloroform is then added and the bottle is shaken vigorously and thencentrifuged at 17 700×g, 15 min, 4° C.

After centrifugation, the aqueous phase is removed and reextracted withME-equilibrated phenol until no interface is obtained. The aqueous phaseis extracted once with chloroform.

The aqueous phase is transferred to a clean centrifuge bottle and 8MLiCl is added to a final concentration of 2M LiCl. RNA is precipitatedovernight at 4° C.

This is then centrifuged for 20 min at 15 300×g, 4° C. The pellet isrinsed with a few ml of 2M LiCl. The pellet is resuspended in 5 ml waterand transferred to a centrifuge tube. 8M LiCl is added to a finalconcentration of 2M LiCl and the RNA is precipitated for 3 hr at 4° C.The RNA is pelleted by centrifugation for 20 min at 12 100×g, 4° C. Thepellet is rinsed with 2M LiCl and resuspended in 2 ml water. 200 μl 3MNa-acetate and 5.5 ml 100% ethanol are added and the RNA precipitated at−20° C. overnight.

The RNA is pelleted for 15 min at 17 700×g, 4° C. The pellet is driedand resuspended in 1 ml water.

The RNA concentration is calculated and purity assessed in a 1:100dilution of the stock using the absorbance at 260 nm, 280 nm, 230 nm and320 nm. The pH is adjusted with conc. Na₂HPO₄ to 1 mM before reading.

Approximately 1 μg from each sample is separated on an agarose gel todetermine the extent of detectable DNA contamination and RNAdegradation. The samples are stored at −70° C.

4.1.2 1^(st) Strand cDNA Synthesis

mRNA is isolated from 4 μg total RNA using paramagnetic beads (DYNALA.S., Oslo, Norway) according to the manufacturer's instructions.

20 μl water is added to each sample and the mRNA is eluted from thebeads at 65° C. for 2 min. The supernatant is collected in a new tubeand the elution is repeated. The two eluates are pooled.

1 μl Oligo dT₁₂₋₁₈₎ (500 ng) 5 μl (from ≧1 μg total RNA) mRNA and waterto 12 μl are mixed together. The mixture is heated to 70° C. for 10 min,chilled immediately on ice and centrifuged briefly to remove thecondensed solution from the tube walls.

4 μl First Strand Buffer (250 mM Tris-HCl, pH 8.3, 375 mM KCl and 15 mMMgCl₂), 2 μl 0.1 mM dithiotreitol and 1 μl dNTP mix (10 mM each of dATP,dCTP, dGTP and dTTP) are added. This is mixed gently and incubated for 2min at 37° C. 1 μl (200 U) reverse transcriptase i.e. Superscript isadded and incubated for 1 hr at 37° C. The tubes are then placed on ice.

4.1.3 2^(nd) (Complementary) Strand cDNA Synthesis

91.8 μl water, 32 μl 2^(nd) strand buffer (100 mM Tris-HCl, pH 7.5, 500mM KCl, 25 mM MgCl₂, 0.25 mg/ml Bovine Serum Albumin and 500 mM(NH₄)₂SO₄, 3 μl dNTP mix, 6 μl 0.1M dithiotreitol, 2 μl E. coli DNALigase (10 U/μl), 4 μl E. coli DNA Polymerase (10 U/μl) and 0.7 μl E.coli RNase H (1 U/μl) are added to the samples. This is incubated for 2hr at 16° C. and then 2 μl T4 DNA Polymerase (10 U)/μg is added. This isincubated for a further 5 min at 16° C.

This is then extracted once with 1 vol. of water-saturated phenol andthen once with 0.5 vol. phenol and 0.5 vol. chloroform:isoamylalcohol(39:1). 3M Na-Acetate, pH 5.2 is added to a final concentration of 0.3Mand mixed gently. 2.5 volumes of 96% Ethanol (−20° C.) is added and themixture is centrifuged at 10 000×g, 4° C. for 30 min.

The supernatant is discarded and 100 μl 75% Ethanol (−20° C.) is addedto the pellet. This mixture is centrifuged at 10 000×g, at 4° C. for 5min. The supernatant is discarded and the pellet is maintained at roomtemperature until dry. The pellet is then resuspended in 15 μl water.

4.1.4 Adapter Annealing

5 pmol/μl each of the two TaqI adapters and the two AseI adapters arepre-mixed.

TaqI adapter 1: GACGATGAGTCCGAC TaqI adapter 2: CGGTCAGGACTCATAseI adapter 1: CTCGTAGACTGCGTACC AseI adapter 2: TAGGTACGCAGTCThe mixture is heated for 2 min at 70° C. and cooled down slowly to 30°C.4.1.5 Ligation of Adapters to the cDNA

The two samples of cDNA (prepared according to step 4.1.3, above) aredigested with TaqI and AseI. 15 μl of the sample is mixed with 3 μlbuffer (50 mM Tris-HCl, pH 7.9, 10 mM MgCl₂, 50 mM NaCl, 1 mMDithiotreitol), 0.3 μl Bovine Serum Albumin (10 mg/ml), 1 μl AseI (10 U)and 1 μl TaqI° (10 U). The mixture is incubated at 37° C. for at least 2hr.

The condensate is spun down and incubated at 65° C. for at least 1.5 hr.1 μl AseI adapter (5 pmol), 1 μl TaqI adapter (50 pmol), 4 μl Ligasebuffer (50 mM Tris-HCl, pH 7.8, 10 mM MgCl₂, 10 mM Dithiotreitol and 25μg/ml Bovine Serum Albumin), 3 μl water and 1 μl T4 DNA Ligase (1 U) isadded. This is incubated at 37° C. for 3 hr. The mixture is then dilutedto 500 μl with TE, 8.0 (10 mM Tris, pH 8.0, 1 mM EDTA).

4.1.6 Preamplification

5 μl samples from the previous step are each mixed with 2.5 μlamplification buffer (0.1M Tris-HCl, pH 8.3, 0.5M KCl, 15 mM MgCl₂ and0.1% (w/v) gelatin), 1 μl (5 ng) AseI preamplification primer, 1 μl (30ng) TaqI preamplification primer, 1 μl dNTP mix, 13.5 μl water and 1 μl(1 U) TaqI DNA Polymerase.

AseI preamplification primer: CTCGTAGACTGCGTACCTAATTaqI preamplification primer: GACGATGAGTCCTGACCGAA temperature cycle of 94° C. for 30 sec, 56° C. for 30 sec and 72° C.for 1 min is run for 25 cycles.

4.1.7 Amplification

5 μl of the samples from the preamplification step are each mixed with 1μl AseI primer (5 ng), 1 μl TaqI primer (30 ng), 0.2 μl Taq Polymerase(1 U), 4 μl amplification buffer, 0.4 μl dNTP mix and 8.4 μl water. Theprimers are end labelled with γ³²P-ATP.

AseI amplification primer: GACTGCGTACCTAATNNTaqI amplification primer: GATGAGTCCTGACCGANN(N denotes any of the four deoxynucleotides)

A temperature cycle is run of 0.7° reduction in the first step for thefirst 11 cycles starting at 65° for 30 sec, 72° C. for 1 min and 94° C.for 30 sec. After the initial 11 cycles the cycling is continued for anadditional 23 cycles with 65° C. for 30 sec, 72° C. for 1 min and 94° C.for 30 sec. The samples are stored at 4° C.

4.1.8 Differential Display

A 6% sequencing type polyacrylamide gel is poured and the sequencing gelapparatus set up. The gel is pre-run until it has achieved 55° C.

5 μl of each sample is mixed with 5 μl sequencing sample dye (95%Formamide, 20 mM EDTA, 0.05% Bromophenol Blue and 0.05% Xylene CyanolFF). The samples are heated at 75°-80° C. for 2 min and immediatelyloaded onto the gel. The samples are positioned close to each other onthe gel.

The gel is run at 55° C. until the Xylene Cyanol FF band has eluted out.The gel is dried and an autoradiogram is positioned on top of the gel.The film is exposed overnight in darkness. Both the film and dry gel aremarked to make it possible to obtain an exact fit for subsequentprocedures. The film is developed.

4.1.9 Selecting Differentially Expressed cDNA Clones as Probes

The pattern between the two samples (normal and diseased) are comparedand samples are selected that are clearly present in one of the samplesbut not in the other. The film is positioned precisely on the dried gel.The gel slice corresponding to a selected fragment (which represents atranscript) on the film is cut out and collected in a tube by theaddition of 2 μl water to a tube and 0.5 μl water to each selected gelfragment within the cutting lines and use of a scalpel to scrape thefragment up and transfer the fragment to the tube. Between one and tenfragments from each reaction with a specific primer may be collected inthis way. Since both of the nucleotides marked N on each of theamplification primers may be one of four possible nucleotides there are4⁴ or 256 possible combinations that can be used in the amplification.Since one to 10 fragments may be picked from any individualamplification using only a single primer, in total between 256 and 2560fragments may be picked from a sample using all permutations of theamplification primer.

A temperature cycle is run as before using the unlabelled amplificationprimers except that the concentration of the primers is increased to 1μM and the dNTP mix to 200 μM and 40 cycles are performed.

30 ng of the amplified fragments (probes) are applied to a Hybond Nfilter using a dot blot apparatus. At least two replicas of the filterare produced. The DNA is fixed to the filter by UV crosslinking.

4.1.10 Hybridization of Test Sample cDNA to Filter with Probe cDNA

The second aliquot of leaves from the plants with the diagnosed diseaseand the healthy plants are used to isolate RNA as described previously.First strand cDNA is prepared as described previously with the exceptionthat 5 μl of [³²P]αdCTP (spec.activity=10 mCi/ml) is added instead of 5μl water to create the labelled sample DNA.

The filter is pre-hybridized in a solution of 4.8×SSC (4.2% (w/v) NaCl,2.1% (w/v) Na-Citrate, pH7.0). 1×Denhardt (0.02% (w/v) Ficoll 400, 0.02%(w/v) polyvinylpyrrolidone 40, 0.02% (w/v) Bovine Serum Albumin,Fraction V), 50% (w/v) Formatted, 0.2M Tris-HCl, pH 7.6, 5% (w/v)dextran sulfate, 0.1% Na-dodecylsulfate at 42° C. for at least 2 hr in ahybridization oven. The labelled sample DNA is boiled for 3 min andimmediately added to the prehybridization solution. This is incubated at42° C. in the hybridization oven for at least 6 hr or overnight.

The filter is then washed 2× in 2×SSC, 0.1% SDS, 50° C., 10 min, thenwashed 2× in 1×SSC, 0.1% SDS, 50° C., 15 min, then washed 2× in 0.1×SSC,0.1% SDS, 50° C., 30 min. The film is exposed in an Instant Imager.

4.1.11 Creating the Standard

One of the signals (resulting from binding of one sample to one of theprobes hybridized to the filter) is considered the standard. The ratiofor all the other signals on the filter are determined relative thisstandard signal.

The ratios of the corresponding signals between the plants with thedisease and those without the same disease are compared.

Results

The results are shown in FIG. 1 for 9 selected cDNAs are shown. P1 hasbeen used as the standard. “Healthy” refers to the sample from thehealthy plants and “Ailment” refers to the sample from the plants withthe disease in question.

4.2 Applying the Developed Method on an Undiagnosed Plant

A leaf sample of 2 g is collected from the plant under study andimmediately frozen in two 1 g aliquots. RNA is extracted as previouslydescribed from one of the aliquots.

1^(st) strand cDNA is synthesized as previously described and labelledby including [³²P]αdCTP in the synthesis.

The labelled sample is hybridized to a filter carrying the selected cDNAprobes, washed and the signals detected on an Instant Imager aspreviously described.

The pattern of relative signals from the different probes on the filteris compared with the patterns from the healthy plants and the plantswith the disease and a diagnosis for the presence or absence of thisdisease in the new plant is made.

EXAMPLE 5 Diagnosis of a Disease/Condition in Humans

The diagnosis may be divided in two steps:

-   -   Development of a reproducible expression pattern (fingerprint)        typical for the disease/condition to be diagnosed.        -   Applying the developed method to a sample from a patient and            comparing the expression pattern to patterns developed for            the disease/condition.

5.1 Development of a Reproducible Expression Pattern Typical for theDisease/Condition to be Diagnosed.

5.1.1 Extraction of Total RNA from Blood

10 ml of blood is collected from a patient who has been diagnosed withthe disease of interest and from a patient without this disease butotherwise as similar as possible with regard to age, sex and otherfactors that may influence the result. Each sample is divided into twoaliquots which are immediately frozen in liquid nitrogen.

Using one aliquot of each sample, after thawing the aliquots arecentrifuged at 10 000×g. for 5 min to pellet the cells. The pellets areresuspended in 1 ml Solution A (4M Guanidine thiocyanate, 25 mMNa-citrate, pH 7.0, 0.5% (w/v) N-laurylsarcosine, 0.1M 2-Mercaptethanol)and the lysate passed through a pipette 7-10 times. 0.1 ml 2 MNa-Acetate, pH 4 is added and mixed thoroughly by inversion. 1 mlwater-saturated phenol is added and mixed thoroughly. 0.2 ml of 49:1chloroform/bromochloropropane is added and mixed thoroughly andincubated for 15 min at 0° C. to 4° C. This mixture is centrifuged for20 min at 10 000×g and the upper aqueous phase is transferred to a cleantube.

The RNA is precipitated by adding 1 ml (1 vol) of 100% isopropanol andthe samples are incubated for 30 min at −20° C., followed bycentrifugation for 10 min at 10 000×g, 4° C.

The RNA pellet is dissolved in 0.3 ml Solution A. The RNA is thenprecipitated with 0.3 ml (1 vol) of 100% isopropanol at −20° C. for 30min. This is centrifuged for 10 min at 10 000×g, 4° C. and thesupernatant is discarded.

The RNA pellet is resuspended in 75% Ethanol, vortexed, and incubatedfor 10-15 min at room temperature. This is centrifuged for 5 min at 10000×g, 4° C., and the supernatant is discarded. The pellet is dried atroom temperature, approximately 10-15 min. The pellet is resuspended in100 μl water and incubated for 10 to 15 min at 55° to 60° C.

The RNA concentration is calculated and purity assessed in a 1:100dilution of the stock using the absorbance at 260 nm, 280 nm, 230 nm and320 nm. The pH is adjusted with conc. Na₂HPO₄ to 1 mM before reading.

Approximately 1 μg from each sample is separated on an agarose gel todetermine the extent of detectable DNA contamination and RNAdegradation. The samples are stored at −70° C.

5.1.2 1^(st) Strand cDNA Synthesis to Selection of DifferentiallyExpressed cDNA Clones as Probes

These steps are performed as described in paragraphs 4.1.2 to 4.1.9above.

5.1.3 Hybridization of Test Sample cDNA to Filter with Probe cDNA

The second aliquot of blood from the normal and diseased patients isused to isolate RNA, prepare labelled first strand cDNA, performhybridization to the filter carrying the probes and expose the film asdescribed in paragraph 4.1.10.

5.1.4 Creating the Standard

The results are standardized relative to a single signal as described in4.1.11 above.

The ratios of the corresponding signals between the patient with thedisease and the patient without the same disease are compared.

Results

Similar results are obtained to those illustrated in FIG. 1.

5.2 Applying the Developed Method on an Undiagnosed Patient.

A blood sample of 2 ml is collected from the patient and immediatelyfrozen in two 1 ml aliquots. RNA is extracted as previously describedfrom one of the aliquots after thawing.

1^(st) strand cDNA is synthesized as previously described and labelledby including [³²P]αdCTP in the synthesis.

The labelled sample is hybridized to a filter carrying the selected cDNAprobes, washed and the signals detected on an Instant Imager aspreviously described.

The pattern of relative signals from the different probes on the filteris compared with the patterns from the healthy patient and the patientwith the disease and a diagnosis for the presence or absence of thisdisease in the new patient is made.

EXAMPLE 6 Different Relative Expression Pattern of 7 Selected ProbesFound in Roots of Norway Spruce (Picea abies) Challenged with DifferentTypes of Stress Methods

1. Seven different informative cDNA probes were attached to a nylonmembrane. Most of the clones were isolated from a subtracted cDNAlibrary of Norway spruce roots infected with the fungal pathogen Pythiumdimorphum (in accordance with the method described in Example 4),whereas others were isolated by similar methods from Norway sprucechallenged with other types of stress. The subtracted library wasprepared according to the method described in Lönneborg et al. (1995),PCR Meth. Applic., 4, pS168-S177. It was known that all of these cloneswere induced by at least one type of stress.2. Norway spruce were grown for 2 months in a growth chamber with aday/night cycle of 12 hr/12 hr light intensity of 500 μmoles×m⁻²×sec⁻¹,watering with optimal nutrition once every day. The plants were thenchallenged either with the nutrition as before (control), maltose media,24 mg (fresh weight) Pythium dimorphum, or 24 mg (fresh weight)Rhizoctonia sp. The plants were challenged for 4 and 8 days. Afterchallenge the roots and upper part were immediately frozen.3. The remaining steps 4 to 10 were performed essentially as describedin Example 4.4. RNA was extracted from the roots and upper parts and checked forquality and yield.5. First and second strand cDNA was synthesized using a polyT primer anda primer based on the presence of a 5′ cap. In this way only full-lengthclones were synthesized avoiding the risk of biased synthesis due tomultiple priming sites.6. All cDNA was amplified using the PCR technique and using the same setof primers.7. The cDNA pool was labelled using the polyT primer.8. Filters, each carrying the 7 probes, were pre-hybridized at 65° C.for 6 hours and the labelled cDNA samples were added to one filter eachand the immobilized filters were allowed to hybridize overnight at 65°C.9. The filters were washed to remove any non-specifically hybridizedlabel.10. The radioactive signals of the individual probes on each filter werecounted using an Instant Imager and the relative signals calculated.

Results

The pattern created for the filters with roots from control (day 4),maltose (day 4 and 8), Pythium dimorphum (day 4 and 8), Rhizotonia sp.(day 4) and needles from control (day 4) and Pythium dimorphum (day 4)are presented in FIG. 2. For maltose at day 4, two independentduplicates were performed which indicated the reproducibility of theexperiment.

The results show that each of these challenges creates distinct patternsusing this set of probes. It can also be seen that the stresses inducethe plants systemically allowing use of different parts of the plantsfor assay. Furthermore, the differential expression of particulartranscripts at different stages of the stress may also be seen.

1-17. (canceled)
 18. A method of obtaining isolated selected mRNAspecies or isolated selected cDNA species useful for diagnosing oridentifying schizophrenia or a stage thereof in a human comprising thesteps of: (a) isolating mRNA from cells from blood of more than onehuman who are known to have schizophrenia or a stage thereof(schizophrenia sample), wherein the resulting isolated mRNA isoptionally subjected to reverse transcription to obtain isolated cDNA;(b) isolating mRNA from corresponding cells from blood of more than onecorresponding normal human (normal sample), wherein the resultingisolated mRNA is optionally subjected to reverse transcription to obtainisolated cDNA; (c) separating mRNA species or cDNA species presentwithin each of the resulting isolated mRNA or isolated cDNA of step (a)and (b), wherein the resulting separated mRNA species are optionallysubject to reverse transcription to obtain separated cDNA species; (d)selecting two or more mRNA species or two or more cDNA species from theresulting separated mRNA species or resulting separated cDNA speciesobtained in step (c), respectively, which are present at a differentlevel in the normal sample than in the schizophrenia sample byidentifying a signal corresponding to each mRNA species or cDNA species,wherein the resulting selected two or more mRNA species are optionallysubjected to reverse transcription to obtain two or more selected cDNAspecies; and (e) isolating the resulting two or more selected mRNAspecies or resulting two or more selected cDNA species obtained in step(d) to obtain isolated selected mRNA species or isolated selected cDNAspecies, wherein the resulting isolated selected mRNA species areoptionally subjected to reverse transcription to obtain isolatedselected cDNA species.
 19. A method of preparing a gene transcriptpattern probe kit comprising the steps of: (a) isolating mRNA from cellsfrom blood of more than one human who are known to have schizophrenia ora stage thereof (schizophrenia sample), wherein the resulting isolatedmRNA is optionally subjected to reverse transcription to obtain isolatedcDNA; (b) isolating mRNA from corresponding cells from blood of morethan one corresponding normal humans (normal sample), wherein theresulting isolated mRNA is optionally subjected to reverse transcriptionto obtain isolated cDNA; (c) separating mRNA species or cDNA speciespresent within each of the resulting isolated mRNA or isolated cDNA ofstep (a) and (b), wherein the resulting separated mRNA species areoptionally subject to reverse transcription to obtain separated cDNAspecies; (d) selecting two or more mRNA species or two or more cDNAspecies from the resulting separated mRNA species or resulting separatedcDNA species obtained in step (c), respectively, which are present at adifferent level in the normal sample than in the schizophrenia sample byidentifying a signal corresponding to each mRNA species or cDNA species,wherein the resulting selected two or more mRNA species are optionallysubjected to reverse transcription to obtain two or more selected cDNAspecies; (e) isolating the resulting two or more selected mRNA speciesor resulting two or more selected cDNA species obtained in step (d) toobtain isolated selected mRNA species or isolated selected cDNA species,wherein the resulting isolated selected mRNA species are optionallysubjected to reverse transcription to obtain isolated selected cDNAspecies; and (f) preparing at least one solid support carrying theresulting isolated selected mRNA species or isolated selected cDNAspecies of step (e) so as to form a gene transcript pattern probe kit.20. The method as claimed in claim 19, wherein said separation in step(c) is performed by a non-sequence based separation technique.
 21. Themethod as claimed in claim 19, wherein in steps (a) and (b), theresulting isolated mRNA is subjected to reverse transcription to obtainisolated cDNA.
 22. The method as claimed in claim 21, where saidisolated cDNA is amplified.
 23. The method as claimed in claim 19,wherein in step (e), between 50 and 100 mRNA species or cDNA species areisolated and selected.
 24. The method as claimed in claim 19, wherein,in step (e), between 10 and 500 mRNA species or cDNA species areisolated and selected.
 25. The method as claimed in claim 19, wherein,in step (c), said separation technique is gel electrophoresis.
 26. Themethod as claimed in claim 19, wherein, prior to step (f), the resultingisolated selected mRNA species or isolated selected cDNA species of step(e) are amplified.
 27. A method of preparing a standard gene transcriptpattern characteristic of schizophrenia or a stage thereof of a humancomprising the steps of: (a) isolating mRNA from cells from blood ofmore than one human who are known to have schizophrenia or a stagethereof (schizophrenia sample), wherein the resulting isolated mRNA isoptionally subjected to reverse transcription to obtain isolated cDNA,(b) hybridizing the resulting isolated mRNA or isolated cDNA of step (a)to two or more mRNA species which are present at a different level incells in a blood sample from more than one normal human than incorresponding cells in a blood sample from more than one humans known tohave schizophrenia or a stage thereof, or to two or more cDNA speciestranscribed from said mRNA species, wherein the two or more mRNA speciesor cDNA species are specific for schizophrenia or a stage thereof,wherein said mRNA or cDNA species are carried on a solid support; and(c) assessing the amount of hybridization so as to obtain said standardgene transcript pattern.
 28. A method of preparing a human test genetranscript pattern for schizophrenia or a stage thereof comprising thesteps of: (a) isolating mRNA from cells from blood of a test human,wherein the resulting isolated mRNA is optionally subjected to reversetranscription to obtain isolated cDNA, (b) hybridizing the resultingisolated mRNA or isolated cDNA of step (a) to two or more mRNA specieswhich are present at a different level in cells in a blood sample frommore then one normal human than in corresponding cells in a blood samplefrom more than one human known to have schizophrenia or a stage thereof,or to two or more cDNA species transcribed from said mRNA species,wherein the two or more mRNA species or cDNA species are specific forschizophrenia or a stage thereof, wherein said mRNA or cDNA species arecarried on a solid support; and (c) assessing the amount ofhybridization so as to obtain said test gene transcript pattern.
 29. Amethod of diagnosing or identifying schizophrenia or a stage thereof ina test human comprising the steps of: (a) isolating mRNA from cells fromblood of a test human, wherein the resulting isolated mRNA is optionallysubjected to reverse transcription to obtain isolated cDNA; (b)hybridizing the resulting isolated mRNA or isolated cDNA of step (a) totwo or more mRNA species which are present at a different level in cellsin a blood sample from more than one normal human than in correspondingcells in a blood sample from more than one human known to haveschizophrenia or a stage thereof, or to two or more cDNA speciestranscribed from said mRNA species, wherein the two or more mRNA speciesor cDNA species are specific for schizophrenia or a stage thereof,wherein said mRNA or cDNA species are carried on a solid support; (c)assessing the amount of hybridization so as to obtain a hybridizationpattern; and (d) comparing the resulting hybridization pattern obtainedin step (c) with a hybridization pattern obtained by hybridizingisolated mRNA or isolated cDNA prepared from corresponding cells fromblood of more than one corresponding human known to schizophrenia or astage thereof to the two or more mRNA species or two or more cDNAspecies transcribed from said mRNA species, wherein said mRNA species orcDNA species are carried on a solid support, so as to determine thedegree of correlation indicative of the presence of schizophrenia or astage thereof, and so as to diagnose or identify schizophrenia or astage thereof in said test human.
 30. The method as claimed in claim 29,wherein in step (a), the resulting isolated mRNA is subjected to reversetranscription to obtain isolated cDNA.
 31. The method as claimed inclaim 30, where said isolated cDNA is amplified.
 32. The method asclaimed in claim 29, wherein when isolated cDNA is obtained, any of saidisolated cDNA is labeled.
 33. The method as claimed in claim 29, whereinin step (b), between 50 and 100 mRNA species or cDNA species are used.34. The method as claimed in claim 29, wherein, in step (b), between 10and 500 mRNA species or cDNA species are used.
 35. The method as claimedin claim 29, wherein said solid support is a filter.
 36. The method asclaimed in claim 29, wherein said method is used to detect a human'sreaction to drug treatment during the treatment of schizophrenia.